Methods for preparing nucleic acid samples

ABSTRACT

In one aspect the present invention provides methods of synthesizing a preparation of nucleic acid molecules, the methods comprising the steps of: (a) utilizing an RNA template to enzymatically synthesize a first DNA molecule that is complementary to at least 50 contiguous bases of the RNA template; (b) utilizing the first DNA molecule as a template to enzymatically synthesize a second DNA molecule, thereby forming a double-stranded DNA molecule wherein the first DNA molecule is hybridized to the second DNA molecule; (c) utilizing the first or second DNA molecule of the double-stranded DNA molecule as a template to enzymatically synthesize a first RNA molecule that is complementary to either the first DNA molecule or to the second DNA molecule; and (d) utilizing the first RNA molecule as a template to enzymatically synthesize a third DNA molecule that is complementary to the first RNA molecule. In another aspect, the present invention provides processed DNA samples prepared by a method of the invention for synthesizing a preparation of nucleic acid molecules. In another aspect, the present invention provides methods for hybridizing a processed DNA sample to a population of immobilized nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of Provisional Application No.60/316,648, filed Aug. 31, 2001.

FIELD OF THE INVENTION

The present invention relates to methods for preparing nucleic acidsamples that are useful for screening populations of immobilized nucleicacid molecules, such as DNA molecules immobilized on a DNA microchip.

BACKGROUND OF THE INVENTION

The characterization of cellular gene expression finds application in avariety of disciplines, such as in the analysis of differentialexpression between different tissue types, different stages of cellulargrowth or between normal and diseased states. Recently, changes in geneexpression have also been used to assess the activity of new drugcandidates and to identify new targets for drug development. The latterobjective is accomplished by correlating the expression of a gene orgenes known to be affected by a particular drug with the expressionprofile of other genes of unknown function when exposed to that samedrug. Genes of unknown function that exhibit the same pattern ofregulation, or signature, in response to the drug are likely torepresent novel targets for pharmaceutical development.

DNA arrays are particularly useful in gene expression analysis at thelevel of transcription (see, e.g., Ramsay, Nature Biotechnol. 16:40-44,1998; Marshall and Hodgson, Nature Biotechnol. 16:27-31, 1998; Lashkariet al., Proc. Natl. Acad. Sci. (USA) 94:130-157, 1997; DeRisi et al.,Science 278:680-6, 1997). In such analysis, the identity and abundanceof a selected nucleic acid sequence in a sample is determined bymeasuring the level of hybridization of the nucleic acid sequence toprobes on the DNA array that comprise complementary sequences. Theselected nucleic acid sequence in a sample can be an mRNA, or a nucleicacid molecule derived from an mRNA that has a nucleic acid sequence thatis identical to, or complementary to, all, or a portion, of the mRNA.Using DNA array expression assays, complex mixtures of labeled nucleicacids (e.g., mRNAs, or nucleic acid molecules derived from mRNAs) can beanalyzed.

The nucleic acid molecules used to screen a DNA array should berepresentative of the mRNA population from which they are derived. All,or substantially all, of the sequences in the mRNA population should berepresented in the nucleic acid molecule population used to screen theDNA array. For example, all portions of individual mRNA molecules shouldbe equally represented in the nucleic acid molecule population used toscreen the DNA array. In this regard, the use of oligo-dT primers, thathybridize to the polyA tail of mRNA molecules, to prime the enzymaticsynthesis of complementary DNA molecules, results in theunderrepresentation of the 3′ ends of long mRNA molecules in thepopulation of complementary DNA molecules.

A proposed solution to this problem is to use a population ofoligonucleotides, having random nucleic acid sequences, to prime theenzymatic synthesis of DNA molecules complementary to the template mRNAmolecules. It is statistically likely that at least one of the randomoligonucleotides will hybridize to at least one portion of each mRNAmolecule in a population, thereby yielding a population of complementaryDNA molecules that represent all, or substantially all, portions of all,or substantially all, mRNA molecules in the template population. Adrawback to this approach, however, is that there is little or noamplification of the sequences in the template mRNA population, therebylimiting the practical usefulness of the technique, for example toproduce enough probe to screen numerous DNA arrays.

Further, the nucleic acid molecules used to screen a DNA array shouldselectively hybridize to complementary nucleic acid molecules, and nothybridize, to a significant extent, to non-complementary nucleic acidmolecules, immobilized on the DNA array. In this regard, the presentinventors have observed that RNA molecules are typically more prone tohybridize to complementary nucleic acid molecules, immobilized on a DNAarray, than are DNA molecules.

Thus, there is a need for methods for synthesizing DNA molecules frommRNA template molecules, wherein: (a) the synthesized DNA moleculesrepresent all, or substantially all, portions of all, or substantiallyall, template mRNA molecules; (b) the abundance of each template mRNAmolecule, and each portion of each template mRNA molecule, is identical,or substantially identical, to the abundance of the identical, orcomplementary, DNA sequence in the population of synthesized DNAmolecules; and (c) the synthetic method is capable of amplifying a smallamount of template mRNA (e.g., 1 μg or less) to yield sufficient probeto screen numerous DNA microarrays. Preferably, the synthesized DNAmolecules selectively hybridize to complementary nucleic acid molecules,and do not hybridize, to a significant extent, to non-complementarynucleic acid molecules immobilized on a DNA array. Moreover, it isdesirable that the synthetic methods controllably yield a population ofDNA molecules wherein all, or substantially all, of the DNA moleculesare complementary to either the sequences of the template mRNAmolecules, or to the complementary sequences of the template mRNAmolecules.

In one aspect, the present invention provides processed nucleic acidsamples that meet the foregoing requirements, and methods for makingsuch processed nucleic acid samples.

SUMMARY OF THE INVENTION

In accordance with the foregoing, in one aspect the present inventionprovides methods of synthesizing a preparation of nucleic acidmolecules, the methods comprising the steps of: (a) utilizing an RNAtemplate to enzymatically synthesize a first DNA molecule that iscomplementary to at least 50 contiguous bases of the RNA template; (b)utilizing the first DNA molecule as a template to enzymaticallysynthesize a second DNA molecule, thereby forming a double-stranded DNAmolecule wherein the first DNA molecule is hybridized to the second DNAmolecule; (c) utilizing the first or second DNA molecule of thedouble-stranded DNA molecule as a template to enzymatically synthesize afirst RNA molecule that is complementary to either the first DNAmolecule or to the second DNA molecule; and (d) utilizing the first RNAmolecule as a template to enzymatically synthesize a third DNA moleculethat is complementary to the first RNA molecule. The double-stranded DNAmolecule prepared in accordance with step (b) can optionally beenzymatically amplified before utilizing the first, or second, DNAmolecule of the double-stranded DNA molecule as a template toenzymatically synthesize a first RNA molecule. The third DNA moleculeprepared in accordance with the methods of this aspect of the inventioncan be labeled with a dye. In some embodiments of the methods of thisaspect of the invention, the third DNA molecule is labeled via anaminoallyl linkage. The methods of this aspect of the invention yieldspopulations of third DNA molecules that are representative of thepopulation of RNA template molecules used to synthesize the third DNAmolecules. In particular, substantially all of the RNA molecules in thepopulation of template RNA molecules are represented in the populationof third DNA molecules, and there is substantially no 5′ or 3′ biaswithin the population of third DNA molecules. The embodiments of themethods of this aspect of the invention that include amplification ofthe double-stranded DNA molecules typically yield an amount of third DNAmolecules that is at least a thousand-fold greater than the amount oftemplate RNA molecules.

The methods of the invention for synthesizing a preparation of nucleicacid molecules are useful in any situation where it is desired tosynthesize a preparation of nucleic acid molecules, such as DNAmolecules, beginning with an RNA template. For example, the methods ofthis aspect of the invention are useful for synthesizing a population ofthird DNA molecules that is used to hybridize to a population ofimmobilized nucleic acid molecules, such as a population of DNAmolecules immobilized on a DNA microarray. For example, third DNAmolecules prepared in accordance with the methods of this aspect of theinvention can be used to hybridize to a DNA chip in order to generate agene expression profile. Gene expression profiling can be done, forexample, for purposes of screening, diagnosis, staging a disease, andmonitoring response to therapy, as well as for identifying genetictargets of drugs and of pathogens.

In another aspect, the present invention provides processed DNA samplesprepared by a method of the invention for synthesizing a preparation ofnucleic acid molecules. The processed DNA samples of the invention canbe utilized in any experiment, process or therapeutic treatment thatrequires DNA. For example, the processed DNA samples of the inventioncan be hybridized to a population of immobilized nucleic acid molecules,such as to a population of DNA molecules immobilized on a Southern blot,to a population of RNA molecules immobilized on a Northern blot, or to apopulation of DNA molecules immobilized on a DNA microarray (such as forthe purpose of gene expression profiling). For example, the processedDNA samples of the invention can be used in the gene expressionprofiling method set forth in Hughes, T. R., et al., NatureBiotechnology 19:342-347 (2001), which publication is incorporatedherein by reference. When used as probes to hybridize to a population ofimmobilized nucleic acid molecules, such as a population of nucleic acidmolecules immobilized on a DNA array, processed DNA samples of theinvention exhibit a high level of hybridization specificity andsensitivity.

In another aspect, the present invention provides methods forhybridizing a processed DNA sample to a population of immobilizednucleic acid molecules, the methods each comprising the step ofhybridizing a processed DNA sample to a population of immobilizednucleic acid molecules, wherein the processed DNA sample is prepared bya method of the invention for synthesizing a preparation of nucleic acidmolecules. The methods of the invention for hybridizing a processed DNAsample to a population of immobilized nucleic acid molecules are usefulin any hybridization experiment wherein DNA molecules are hybridized toa population of immobilized nucleic acid molecules, such as a populationof DNA molecules immobilized on a DNA micro array.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a representative embodiment of the methods of the inventionfor synthesizing a preparation of nucleic acid molecules.

FIG. 2 shows a representative embodiment of the methods of theinvention, and also shows the amplification efficiency of each step ofthe representative embodiment. The abbreviations are: RT, reversetranscriptase; mRNA, messenger RNA; cDNA. complementary DNA; dsDNA,double-stranded DNA; cRNA, complementary RNA; IVT, in vitrotranscription. The term “coupling” refers to coupling the DNA to Cy dyemolecules.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention.

In one aspect, the present invention provides methods of synthesizing apreparation of nucleic acid molecules, the methods comprising the stepsof: (a) utilizing an RNA template to enzymatically synthesize a firstDNA molecule that is complementary to at least 50 contiguous bases ofsaid RNA template; (b) utilizing the first DNA molecule as a template toenzymatically synthesize a second DNA molecule, thereby forming adouble-stranded DNA molecule wherein the first DNA molecule ishybridized to the second DNA molecule; (c) utilizing the first or secondDNA molecule of the double-stranded DNA molecule as a template toenzymatically synthesize a first RNA molecule that is complementary toeither the first DNA molecule or to the second DNA molecule; and (d)utilizing the first RNA molecule as a template to enzymaticallysynthesize a third DNA molecule that is complementary to the first RNAmolecule. The double-stranded DNA molecule prepared in accordance withstep (b) can optionally be enzymatically amplified before utilizing thefirst, or second, DNA molecule of the double-stranded DNA molecule as atemplate to enzymatically synthesize a first RNA molecule. The third DNAmolecule prepared in accordance with the methods of this aspect of theinvention can be labeled with a dye. In some embodiments of the methodsof this aspect of the invention, the third DNA molecule is labeled viaan aminoallyl linkage.

Preparation of RNA molecules useful as templates. RNA molecules usefulas templates in the methods of this aspect of the invention can beisolated from any organism or part thereof, including organs, tissues,and/or individual cells. Any suitable RNA preparation can be utilized,such as total cellular RNA, or such as cytoplasmic RNA or such as an RNApreparation that is enriched for messenger RNA (mRNA), such as RNApreparations that include greater than 70%, or greater than 80%, orgreater than 90%, or greater than 95%, or greater than 99% messengerRNA. Typically, RNA preparations that are enriched for messenger RNA areutilized to provide the RNA template in the practice of the methods ofthis aspect of the invention. Messenger RNA can be purified inaccordance with any art-recognized method, such as by the use ofoligo-dT columns (see, e.g., Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1, Chapter 7, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

RNA may be isolated from eukaryotic cells by procedures that involvelysis of the cells and denaturation of the proteins contained therein.Cells of interest include wild-type cells, drug-exposed wild-type cells,modified cells, and drug-exposed modified cells.

Additional steps may be employed to remove DNA. Cell lysis may beaccomplished with a nonionic detergent, followed by microcentrifugationto remove the nuclei and hence the bulk of the cellular DNA. In oneembodiment, RNA is extracted from cells of the various types of interestusing guanidinium thiocyanate lysis followed by CsCl centrifugation toseparate the RNA from DNA (Chirgwin et al., 1979, Biochemistry18:5294-5299). Messenger RNA is selected by selection with oligo-dTcellulose (see Sambrook et al., supra). Separation of RNA from DNA canalso be accomplished by organic extraction, for example, with hot phenolor phenol/chloroform/isoamyl alcohol.

If desired, RNase inhibitors may be added to the lysis buffer. Likewise,for certain cell types, it may be desirable to add a proteindenaturation/digestion step to the protocol.

The sample of RNA can comprise a plurality of different mRNA molecules,each different mRNA molecule having a different nucleotide sequence. Ina specific embodiment, the mRNA molecules in the RNA sample comprise atleast 100 different nucleotide sequences. In other embodiments, the mRNAmolecules of the RNA sample comprise at least 500, 1,000, 5,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 90,000 or 100,000different nucleotide sequences. In another specific embodiment, the RNAsample is a mammalian RNA sample, the mRNA molecules of the mammalianRNA sample comprising about 20,000 to 30,000 different nucleotidesequences.

Synthesis of first DNA molecules. In the practice of the methods of thisaspect of the invention, DNA molecules, referred to herein as first DNAmolecules, are synthesized that are complementary to the RNA templatemolecules. Individual first DNA molecules can be complementary to awhole RNA template molecule, or to a portion thereof. For example, afirst DNA molecule can be complementary to the portion of a template RNAmolecule that extends from the 3′ end of the template RNA molecule tothe midpoint of the template RNA molecule; similarly, by way of example,a first DNA molecule can be complementary to the portion of an RNAtemplate molecule that extends from the 5′ end of the RNA molecule tothe midpoint of the RNA molecule. Many first DNA molecules arecomplementary to at least 50 contiguous bases of an RNA templatemolecule. Thus, in this example, the complete complementary sequence ofa RNA template molecule is represented in the population of first DNAmolecules; the 5′ half of the complementary sequence is represented onone DNA molecule, and the 3′ half of the complementary sequence isrepresented on another DNA molecule.

Thus, in the practice of the methods of this aspect of the invention, apopulation of first DNA molecules is synthesized that includesindividual DNA molecules that are each complementary to all, or to aportion, of a template RNA molecule. Typically, at least a portion ofthe complementary sequence of at least 95% (more typically at least 99%)of the template RNA molecules are represented in the population of firstDNA molecules. Of the template RNA molecules that are represented in thepopulation of first DNA molecules, typically at least 95% (moretypically at least 98%) of the complementary sequence of eachrepresented template RNA molecule is present in the population of firstDNA molecules.

Any reverse transcriptase molecule can be utilized to synthesize thefirst DNA molecules, such as those derived from Moloney murine leukemiavirus (MMLV-RT), avian myeloblastosis virus (AMV-RT), bovine leukemiavirus (BLV-RT), Rous sarcoma virus (RSV) and human immunodeficiencyvirus (HIV-RT). A reverse transcriptase lacking RNaseH activity (e.g.,SUPERSCRIPT II™ sold by Stratagene, La Jolla, Calif.) is preferred,however, because, in the absence of an RNaseH activity, synthesis of thesecond DNA molecules does not occur during synthesis of the first DNAmolecules, thereby preventing incorporation of the first primersequence(s) into the second DNA molecules. The reverse transcriptasemolecule should also preferably be thermostable so that the first strandsynthesis reaction can be conducted at as high a temperature aspossible, while still permitting hybridization of the first primer(s) tothe RNA template molecules. In some embodiments of the methods of thisaspect of the invention, in order to minimize the amount ofdouble-stranded DNA synthesized during the synthesis of the first DNAmolecules, a reverse transcriptase that lacks RNase H activity isutilized, and the duration of the first DNA molecules synthesis reactionis less than two hours (such as between 20 minutes and two hours, orbetween 15 minutes and 25 minutes).

Priming the synthesis of the first DNA molecules. The synthesis of thefirst DNA molecules is primed using any suitable primer, typically anoligonucleotide in the range of ten to 60 bases in length. The nucleicacid sequence of the oligonucleotide used to prime the synthesis of thefirst DNA molecules is therefore incorporated into the sequence of each,synthesized, first DNA molecule. Oligonucleotides that are useful forpriming the synthesis of the first DNA molecules can hybridize to anyportion of the RNA template molecules, including the oligo-dT tail. Insome embodiments, the synthesis of the first DNA molecules is primedusing a first primer mixture comprising a multiplicity of first primers,wherein each of the first primers includes a random sequence portion,and a defined sequence portion. The random sequence portion comprises arandom sequence of nucleic acid residues. Statistically, it is likelythat most, or all, of the random sequences are sufficientlycomplementary to a portion of one or more RNA template molecules, to beable to hybridize thereto under the conditions utilized to hydridize thefirst primer molecules to the RNA template molecules. The randomsequence portion typically consists of from four to 20 nucleic acidresidues, such as from four to 15 nucleic acid residues, such as fromsix to nine nucleic acid residues. In one embodiment, the randomsequence portion consists of nine nucleic acid residues. Typically, thedefined sequence portion is located 5′ to the random sequence portion.

The defined sequence portion of the first primers comprises a knownsequence of nucleic acid residues, and can include an RNA polymerasepromoter. The RNA polymerase promoter sequence is therefore incorporatedinto the sequence of the first DNA molecules, which can thereafter beutilized as templates for the synthesis of RNA molecules that arecomplementary in sequence to the first DNA molecules. Any RNA polymerasepromoter sequence can be included in the defined sequence portion of thefirst primers. Representative examples of useful RNA polymerasepromoters include a T7 RNA polymerase promoter and an SP6 RNA polymerasepromoter. A representative defined sequence portion of a first primerthat includes a T7 RNA polymerase promoter sequence is 5′ ACTA TAG GGAGA 3′ (SEQ ID NO:1), which is the defined sequence portion ofrepresentative first primer molecule ShT7N9 5′ ACTA TAG GGA GAN NNN NNNNN 3′ (SEQ ID NO:2).

The nucleic acid sequence of an exemplary primer useful for priming thesynthesis of the first DNA molecule, and which does not include an RNApolymerase promoter sequence, is 5′ TAG ATG CTG TTG NNN NNN NNN 3′ (SEQID NO:3), which is called primer ShDNP256. The defined sequence portionof ShDNP256 is 5′ TAG ATG CTG TTG 3′ (SEQ ID NO:4).

In some embodiments, the synthesis of the first DNA molecules is primedusing a mixture of primers, wherein the mixture includes poly-dT primersthat each comprise a poly-dT portion and a defined sequence portion,wherein the poly-dT portion is located 5′ to the defined sequenceportion. The poly-dT portion of each poly-dT primer typically consistsof from five to 25 nucleic acid residues, such as from 15-25 nucleicacid residues, such as 18 nucleic acid residues. In some embodiments,the poly-dT primers are used with a first primer mixture comprising amultiplicity of first primers, wherein each of the first primerscomprises a random sequence portion and a defined sequence portion. Thenucleic acid sequence of the defined sequence portion of the poly-dTprimer is typically identical to the nucleic acid sequence of thedefined sequence portion of the primers of the first primer mixture. Inthis way, every first DNA molecule includes the same defined sequenceportion which can subsequently be utilized, for example, as ahybridization target for a primer that primes the synthesis of acomplementary DNA molecule, or, for example, as an RNA polymerasepromoter. Thus, in some embodiments, the defined sequence portion of thepoly-dT primer comprises an RNA polymerase promoter, such as a T7 RNApolymerase promoter, such as the T7 RNA polymerase promoter having thenucleic acid sequence set forth in SEQ ID NO:1.

Hybridization of oligonucleotide primers. The following remarks describeconditions for hybridizing oligonucleotide primers to target nucleicacid molecules, such as hybridizing first primers to mRNA molecules inthe practice of the synthetic methods of the invention.

Typically, for oligonucleotide molecules less than 100 bases in length,hybridization conditions are 5 to 10° C. below the homoduplex meltingtemperature (Tm); see generally, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, 1987; Ausubel etal., Current Protocols in Molecular Biology, Greene Publishing, 1987).

Preparing oligonucleotides useful as primers. The following remarksdescribe representative methods and compositions useful for making anyoligonucleotide primer utilized in the practice of the presentinvention, including oligonucleotides useful for priming synthesis ofthe first DNA molecules.

A primer may be prepared by any suitable method, such as phosphotriesterand phosphodiester methods of synthesis, or automated embodimentsthereof. It is also possible to use a primer that has been isolated froma biological source, such as a restriction endonuclease digest.

An oligonucleotide primer can be DNA, RNA, chimeric mixtures orderivatives or modified versions thereof, so long as it is still capableof priming the desired reaction. The oligonucleotide primer can bemodified at the base moiety, sugar moiety, or phosphate backbone, andmay include other appending groups or labels, so long as it is stillcapable of priming the desired amplification reaction.

For example, an oligonucleotide primer may comprise at least onemodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5N-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

In another embodiment, the oligonucleotide primer comprises at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the oligonucleotide primer comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or analog thereof.

An oligonucleotide primer for use in the methods of the invention may bederived by cleavage of a larger nucleic acid fragment using non-specificnucleic acid cleaving chemicals or enzymes or site-specific restrictionendonucleases; or by synthesis by standard methods known in the art,e.g., by use of an automated DNA synthesizer (such as are commerciallyavailable from Biosearch, Applied Biosystems, etc.) and standardphosphoramidite chemistry. As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al. (Nucl.Acids Res. 16:3209-3221, 1988), methylphosphonate oligonucleotides canbe prepared by use of controlled pore glass polymer supports (Sarin etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451).

Once the desired oligonucleotide is synthesized, it is cleaved from thesolid support on which it was synthesized and treated, by methods knownin the art, to remove any protecting groups present. The oligonucleotidemay then be purified by any method known in the art, includingextraction and gel purification. The concentration and purity of theoligonucleotide may be determined by examining oligonucleotide that hasbeen separated on an acrylamide gel, or by measuring the optical densityat 260 nm in a spectrophotometer.

Hydrolysis of template RNA molecules and removal of first primers. Insome embodiments, the RNA template molecules are hydrolyzed, and all, orsubstantially all (typically more than 99%), of the first primers areremoved, after synthesis of the first DNA molecules, and beforesynthesis of the second DNA molecules. Hydrolysis of the RNA templatecan be achieved, for example, by alkalinization of the solutioncontaining the RNA template (e.g., by addition of an aliquot of aconcentrated sodium hydroxide solution). The primers can be removed, forexample, by applying the solution containing the RNA template molecules,first DNA molecules, and the first primers, to a column that separatesnucleic acid molecules on the basis of size. The purified, first DNAmolecules, can then, for example, be precipitated and redissolved in asuitable buffer for the next step of the methods of this aspect of theinvention.

Synthesis of the second DNA molecules. In the practice of the methods ofthe invention, DNA molecules, referred to herein as second DNAmolecules, are synthesized that are complementary to the first DNAmolecules. Individual second DNA molecules can be complementary to awhole first DNA molecule, or to a portion thereof. For example, a secondDNA molecule can be complementary to a portion of a first DNA moleculethat is located between the 3′ end of the first DNA molecule and themidpoint of the first DNA molecule; similarly, by way of example, asecond DNA molecule can be complementary to a portion of a first DNAmolecule that is located between the 5′ end of the first DNA moleculeand the midpoint of the first DNA molecule. Typically, second DNAmolecules are each complementary to at least 50 contiguous bases of afirst DNA molecule.

Thus, in the practice of the methods of this aspect of the invention, apopulation of second DNA molecules is synthesized that includesindividual second DNA molecules that are each complementary to all, orto a portion, of a first DNA molecule. Typically, at least a portion ofthe complementary sequence of at least 95% (more typically at least 98%,such as at least 99%) of the first DNA molecules are represented in thepopulation of second DNA molecules. Of the first DNA moleculecomplementary sequences that are represented in the population of secondDNA molecules, typically at least 95% (more typically at least 98%, suchas at least 99%) of the complementary sequence of each represented firstDNA molecule is present in the population of second DNA molecules.

A DNA-dependent, DNA polymerase is utilized to synthesize the second DNAmolecules. For example, the Klenow fragment of DNA polymerase I can beutilized to synthesize the second DNA molecules. The synthesis of thesecond DNA molecules is primed using any suitable primer or primers,provided that the primer(s) used to prime synthesis of the second DNAmolecules does not have the same nucleic acid sequence as the primer(s)used to prime the synthesis of the first DNA molecules. In this way,both the first and the second DNA molecules include a unique primersequence. The unique primer sequence included in the first DNA moleculesis not included in the second DNA molecules, and the unique primersequence that is included in the second DNA molecules is not included inthe first DNA molecules. Depending on the choice of primer sequence,these unique primer sequences can be used, for example, to selectivelydirect DNA-dependent RNA synthesis from either the first or the secondDNA molecules or, for example, to selectively direct DNA-dependent DNAsynthesis from either the first DNA molecules, or from the second DNAmolecules.

In some embodiments, the synthesis of the second DNA molecules is primedusing a second primer mixture comprising a multiplicity of second primermolecules, wherein each second primer molecule comprises a randomsequence portion and a defined sequence portion. The defined sequenceportion is located 5′ to the random sequence portion. The sequence ofthe defined sequence portion of the second primer molecules is notpresent in the sequence of the first primer molecules. Thus, forexample, the defined sequence portion of the second primer molecules caninclude the nucleic acid sequence of an RNA polymerase promoter, such asa T7 RNA polymerase promoter, such as the sequence of the T7 RNApolymerase promoter set forth in SEQ ID NO:1. A representative nucleicacid sequence of a first primer molecule, called ShT7N9, that includes adefined sequence portion including a T7 promoter sequence is set forthin SEQ ID NO:2.

An example of a primer, that does not include an RNA polymerase promotersequence, useful for priming the synthesis of the second DNA moleculesis set forth in SEQ ID NO:3, which shows the nucleic acid sequence ofprimer ShDNP256.

Thus, in one representative embodiment of the methods of the invention,the defined sequence portion of the first primer mixture includes an RNApolymerase promoter (such as the T7 RNA polymerase sequence set forth inSEQ ID NO:1), and the defined sequence portion of the second primermixture does not include an RNA polymerase promoter sequence. Again, byway of representative example, in one embodiment of the methods of theinvention, the defined sequence portion of the second primer mixtureincludes an RNA polymerase promoter sequence (such as the T7 RNApolymerase promoter sequence set forth in SEQ ID NO:1), and the definedsequence portion of the first primer mixture does not include an RNApolymerase promoter sequence.

Oligonucleotides useful for priming synthesis of the second DNAmolecules can be made using any art-recognized method, such as byutilizing the methods and compositions described herein under theheading “Preparing oligonucleotides useful as primers.”

In some embodiments of the methods of this aspect of the invention, thereaction time for the synthesis of the second DNA molecules is fromabout 45 minutes to about sixty minutes.

Purification of double-stranded DNA molecules. Synthesis of the secondDNA molecules yields a population of double-stranded DNA moleculeswherein the first DNA molecules are hybridized to the second DNAmolecules. Typically, the double-stranded DNA molecules are purified toremove substantially all nucleic acid molecules shorter than 100 basepairs, including all, or substantially all (i.e., typically more than99%), of the second primers. Purification can be achieved by anyart-recognized means, such as by elution through a size-fractionationcolumn. The purified, second DNA molecules can then, for example, beprecipitated and redissolved in a suitable buffer for the next step ofthe methods of this aspect of the invention.

Amplification of the double-stranded DNA molecules. In the practice ofthe methods of this aspect of the invention, either the first DNAmolecules or the second DNA molecules of the double-stranded DNAmolecules are utilized as templates to enzymatically synthesize firstRNA molecules that are complementary in sequence to either the first DNAmolecules or to the second DNA molecules (i.e., complementary to thetemplate DNA molecules). Typically, however, before synthesis of thefirst RNA molecules, the double-stranded DNA molecules are enzymaticallyamplified using the polymerase chain reaction. Any suitable primers canbe used to prime the polymerase chain reaction. Typically, two primersare used; one primer hybridizes to the defined portion of the firstprimer sequence (or to the complement thereof), and the other primerhybridizes to the defined portion of the second primer sequence (or tothe complement thereof).

Thus, for example, the highlighted portion of the T7 primer sequence 5′AAT TAA TAC GAC TCA CTA TAG GGA GA 3′ (SEQ ID NO:5) is identical to thehighlighted portion of the ShT7N9 primer sequence 5′ ACTA TAG GGA GANNNN NNN NN 3′ (SEQ ID NO:2). Under appropriate hybridization conditions,the highlighted portion of the T7 primer (SEQ ID NO:5) will hybridize tothe complement of the highlighted portion of the ShT7N9 primer sequence(SEQ ID NO:2). Thus, in a PCR amplification reaction, the T7 primer (SEQID NO:5) can be used to prime synthesis of a first or second DNAmolecule that includes the complement of the ShT7N9 primer sequence (SEQID NO:2).

Similarly, the highlighted portion of the DP256 primer 5′ GTT CGA GACCTC TAG ATG CTG TTG 3′ (SEQ ID NO:6) is identical to the highlightedportion of the ShDNP256 primer 5′ TAG ATG CTG TTG NNN NNN NNN 3′ (SEQ IDNO:3). Thus, in a PCR amplification reaction, the highlighted portion ofthe DP256 primer (SEQ ID NO:6) can be used to prime synthesis of a firstor second DNA molecule that includes the complement of the highlightedportion of the ShDNP256 primer sequence (SEQ ID NO:3).

In general, the greater the number of amplification cycles during thepolymerase chain reaction, the greater the amount of amplified DNA thatis obtained. On the other hand, too many amplification cycles may resultin randomly biased amplification of the double-stranded DNA. Thus, forexample, if a sample of purified messenger RNA is split into twoidentical portions, and each portion is utilized as template tosynthesize double-stranded DNA in accordance with the methods of theinvention, and too many amplification cycles are utilized during thepolymerase chain reaction step; then the composition of the amplified,double-stranded, DNA derived from the two identical portions of the RNAsample may be significantly different. Thus, in some embodiments, adesirable number of amplification cycles is between one and 25amplification cycles, such as from five to 15 amplification cycles, suchas ten amplification cycles. Where an amplification step is included inthe methods of the invention, the amplified, double-stranded, DNA istypically purified to remove nucleic acid molecules consisting of lessthan about 100 base pairs. By way of example, purification can beachieved by the use of a size-fractionation column.

In some embodiments of the methods of this aspect of the invention, fromabout 100 nanograms (ng) to about 200 ng of the double-stranded DNAmolecules are used as substrate in a PCR amplification reaction.

Synthesis of the first RNA molecules. In the practice of the methods ofthe invention, either the first DNA molecules or the second DNAmolecules of the double-stranded DNA molecules are utilized as templatesto enzymatically synthesize first RNA molecules that are complementaryin sequence to either the first DNA molecules or to the second DNAmolecules (i.e., complementary to the template DNA molecules). The RNAsynthesis reaction is catalyzed by an RNA polymerase. Representativeexamples of useful RNA polymerase molecules include the SP6 RNApolymerase and the T7 RNA polymerase. The first or second DNA moleculesthat are used as the templates for synthesis of the first RNA moleculesincludes an RNA polymerase promoter sequence that was introduced duringsynthesis of the first or second DNA molecules. The RNA polymerasepromoter sequence was included in the sequence of the primer(s) used toprime synthesis of the first or second DNA molecules. For example,primer ShT7N9, having the nucleic acid sequence set forth in SEQ IDNO:2, includes the sequence of a T7 RNA polymerase promoter (SEQ IDNO:1). Thus, if primer ShT7N9 (SEQ ID NO:2) is utilized to prime thesynthesis of the first DNA molecules, then each of the first DNAmolecules includes the sequence of the T7 RNA polymerase promoter (SEQID NO:1) included within primer ShT7N9 (SEQ ID NO:2), and this T7 RNApolymerase promoter (SEQ ID NO:1) can subsequently be utilized topromote the synthesis of a population of RNA molecules that arecomplementary in sequence to the population of first DNA molecules.

The first RNA molecules are typically purified to remove nucleic acidmolecules less than 100 bases long. Purification can be achieved by anyart-recognized means, such as by the use of a size-fractionation column.

In some embodiments of the methods of this aspect of the invention, from400 ng to 600 ng (such as about 500 ng) of amplified, double-stranded,DNA molecules are utilized as template for the synthesis of the firstRNA molecules.

Synthesis of the third DNA molecules. In the practice of the methods ofthe invention, DNA molecules, referred to herein as third DNA molecules,are synthesized that are complementary to the first RNA molecules.Individual third DNA molecules can be complementary to a whole first RNAmolecule, or to a portion thereof. For example, a third DNA molecule canbe complementary to a portion of a first RNA molecule that is locatedbetween the 3′ end of the first RNA molecule and the midpoint of thefirst RNA molecule; similarly, by way of example, a third DNA moleculecan be complementary to a portion of a first RNA molecule that islocated between the 5′ end of the first RNA molecule and the midpoint ofthe first RNA molecule. Typically, third DNA molecules are eachcomplementary to at least 50 contiguous bases of a first RNA molecule.

Thus, in the practice of the methods of this aspect of the invention, apopulation of third DNA molecules is synthesized that includesindividual DNA molecules that are each complementary to all, or to aportion, of a first RNA molecule. Typically, at least a portion of thecomplementary sequence of at least 95% (more typically at least 98%,such as at least 99%) of the first RNA molecules are represented in thepopulation of third DNA molecules. Of the complementary sequences offirst RNA molecules that are represented in the population of third DNAmolecules, typically at least 95% (more typically at least 98%) of thecomplementary sequence of each represented first RNA molecule is presentin the population of third DNA molecules.

The synthesis of the third DNA molecules is catalyzed by a reversetranscriptase molecule, preferably a reverse transcriptase molecule thatdoes not possess an RNAse H enzymatic activity (e.g., SUPERSCRIPT II™),thereby preventing the synthesis of DNA molecules that are complementaryin sequence to the third DNA molecules. The synthesis of the third DNAmolecule can be primed by any suitable primer, or mixture of suitableprimers. In some embodiments, the synthesis of the third DNA molecule isprimed using a population of random primers, wherein substantially allof the random primers consist of a random sequence of nine bases.

In some embodiments of the methods of this aspect of the invention,about 3 μg of first RNA molecules are utilized as template for thesynthesis of the third DNA molecules.

Hydrolysis of the first RNA molecules and removal of primers. In someembodiments, the first RNA molecules are hydrolyzed, and all, orsubstantially all (typically more than 99%), of the primers are removed,after synthesis of the third DNA molecules. Hydrolysis of the first RNAmolecules can be achieved, for example, by alkalinization of thesolution containing the RNA template (e.g., by addition of an aliquot ofa concentrated sodium hydroxide solution). The primers can be removed,for example, by applying the solution containing the first RNAmolecules, third DNA molecules, and the primers, to a column thatseparates nucleic acid molecules on the basis of size. The purified,third DNA molecules, can then be precipitated and redissolved in asuitable buffer for the next step of the methods of this aspect of theinvention.

Labelling the third DNA molecules with a dye. Optionally, the third DNAmolecules can be labeled with a dye molecule to facilitate the detectionof the third DNA molecules when they are used as a probe in ahybridization experiment, such as a probe used to screen a DNA chip. Anysuitable dye molecules can be utilized, provided that they are attachedto the third DNA molecules by aminoallyl linkages. Examples of suitabledyes include fluorophores and chemiluminescers.

By way of example, third DNA molecules can be coupled to dye moleculesvia aminoallyl linkages by incorporating allylamine-derivatizednucleotides (e.g., allylamine-dATP, allylamine-dCTP, allylamine-dGTP,and/or allylamine-dTTP) into the third DNA molecules during synthesis ofthe third DNA molecules. The allylamine-derivatized nucleotide(s) canthen be coupled, via an aminoallyl linkage, to N-hydroxysuccinimideester derivatives (NHS derivatives) of dyes (e.g., Cy-NHS, Cy3-NHSand/or Cy5-NHS). Again by way of example, in another embodiment,dye-labeled nucleotides may be incorporated into the third DNA moleculesduring synthesis of the third DNA molecules, which labels the third DNAmolecules directly.

It is also possible to include a spacer (usually 5-16 carbon atoms long)between the dye and the nucleotide, which may improve enzymaticincorporation of the modified nucleotides during synthesis of the thirdDNA molecules.

Representative examples of useful fluorophores are as follows:

-   -   4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid    -   acridine and derivatives:    -   acridine    -   acridine isothiocyanate    -   5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)    -   4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate        (Lucifer Yellow VS)    -   anthranilamide    -   Brilliant Yellow

coumarin and derivatives:

-   -   coumarin    -   7-amino-4-methylcoumarin (AMC, Coumarin 120)    -   7-amino-4-trifluoromethylcoumarin (Coumarin 151)    -   Cy3    -   Cy5    -   cyanosine    -   4′,6-diaminidino-2-phenylindole (DAPI)    -   5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)    -   7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin        diethylenetriamine pentaacetate    -   4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid    -   4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid    -   5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl        chloride)    -   4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)    -   4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC)

eosin and derivatives:

-   -   eosin    -   eosin isothiocyanate

erythrosin and derivatives:

-   -   erythrosin B    -   erythrosin isothiocyanate    -   ethidium

fluorescein and derivatives:

-   -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF)    -   2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate    -   QFITC (XRITC)    -   fluorescamine    -   IR144    -   IR1446    -   Malachite Green isothiocyanate    -   4-methylumbelliferone    -   ortho cresolphthalein    -   nitrotyrosine    -   pararosaniline    -   Phenol Red    -   B-phycoerythrin    -   o-phthaldialdehyde

pyrene and derivatives:

-   -   pyrene    -   pyrene butyrate    -   succinimidyl 1-pyrene butyrate    -   Reactive Red 4 (Cibacron7 Brilliant Red 3B-A)

rhodamine and derivatives:

-   -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride    -   rhodamine (Rhod)    -   rhodamine B    -   rhodamine 110    -   rhodamine 123    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)    -   N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)    -   tetramethyl rhodamine    -   tetramethyl rhodamine isothiocyanate (TRITC)    -   riboflavin    -   rosolic acid    -   terbium chelate derivatives

Representative method of the invention. FIG. 1 shows a representativemethod of the present invention. In the method disclosed in the FIG. 1,a population of messenger RNA molecules are used as templates tosynthesize first DNA molecules, thereby yielding double-strandedmolecules in which a messenger RNA molecule is hybridized to acomplementary first DNA molecule. The synthesis of first DNA moleculesis catalyzed by reverse transcriptase, and the reaction is primed by afirst primer mixture, which includes a multiplicity of first primermolecules (called ShDNP256) that each have the nucleic acid sequenceshown in SEQ ID NO:3. Each of the first primer molecules (SEQ ID NO:3)includes a defined sequence portion and a random sequence portion. Thedefined sequence portion is located 5′ to the random sequence portion.The nucleic acid sequence of the defined sequence portion is set forthin SEQ ID NO:4. Thus, the nucleic acid sequence set forth in SEQ ID NO:4is incorporated into each first DNA molecule.

The sequence of the random sequence portion of each first primermolecule (SEQ ID NO:3) is different from the sequence of the randomsequence portion of substantially every other first primer molecule (SEQID NO:3). Thus, it is statistically likely that at least one firstprimer molecule (SEQ ID NO:3) will hybridize to every messenger RNAmolecule within the population of messenger RNA molecules. More than onefirst primer molecule (SEQ ID NO:3) may hybridize to a single, template,messenger RNA molecule, thereby permitting the synthesis of more thanone first DNA molecule corresponding to more than one portion of thetemplate messenger RNA molecule. Thus, the complementary sequence ofevery portion, or almost every portion, of every messenger RNA molecule,or almost every messenger RNA molecule, in the population of messengerRNA molecules is represented within the population of first DNAmolecules.

The population of double-stranded nucleic acid molecules, that eachinclude a first DNA molecule hybridized to a messenger RNA templatemolecule, is then hydrolyzed under alkaline conditions to degrade themessenger RNA molecules. The hydrolyzed mixture is then applied to acolumn that separates nucleic acid molecules on the basis of size. Inthis way, nucleic acid molecules of less than 100 base pairs are removedfrom the population of first DNA molecules. The purified first DNAmolecules are then utilized as templates to enzymatically synthesizesecond DNA molecules. The synthesis of the second DNA molecules iscatalyzed by the Klenow fragment of DNA polymerase I, and the synthesisof the second strand DNA molecules is primed using a second primermixture that includes a multiplicity of second primer molecules (calledShT7N9 (SEQ ID NO:2)) which each include a defined sequence portion (SEQID NO:1) and a random sequence portion, wherein the defined sequenceportion (SEQ ID NO:1) is located 5′ to the random sequence portion.

Thus, each second DNA molecule incorporates at least one ShT7N9 definedsequence portion (SEQ ID NO:1). The product of the second DNA moleculesynthesis reaction is a double-stranded DNA molecule in which a secondDNA molecule is hybridized to a complementary first DNA molecule.Nucleic acid molecules of less than 100 bases are separated from thepopulation of double-stranded DNA molecules by elution from a columnthat separates nucleic acid molecules on the basis of size. Theresulting, purified, double-stranded DNA molecules are then amplifiedusing the polymerase chain reaction (PCR). The PCR reaction is catalyzedby the Taq polymerase and the reaction is primed by the T7 primer (SEQID NO:5) and primer DP256 (SEQ ID NO:6), which hybridize to thecomplement of the defined sequence portion (SEQ ID NO:1) of primerShT7N9 (SEQ ID NO:2), and to the complement of the defined sequenceportion (SEQ ID NO:4) of primer ShDNP256 (SEQ ID NO:3), respectively. Inthe embodiment of the methods shown in FIG. 1, the purified,double-stranded, DNA molecules are subjected to ten rounds of PCRamplification. The amplified, double-stranded, DNA molecules are thenpurified to remove nucleic acid molecules of less than about 100 bases.Purification is achieved using a column that separates nucleic acidmolecules on the basis of size.

The second DNA molecules of the purified, amplified, double-stranded DNAmolecules are then utilized as templates to synthesize a population offirst RNA molecules. Each of the first RNA molecules is, therefore,complementary to a second DNA molecule, or to a portion of a second DNAmolecule. The synthesis of the first RNA molecules is catalyzed by T7RNA polymerase that utilizes the T7 promoter, included in the definedsequence portion (SEQ ID NO:1) of primer ShT7N9 (SEQ ID NO:2), that isincorporated into the second DNA molecules during their synthesis. Thefirst RNA molecules are purified by absorption onto a column, thatspecifically absorbs RNA, and elution therefrom.

The purified first RNA molecules are then used as templates tosynthesize a population of third DNA molecules. The synthesis of thethird DNA molecules is catalyzed by reverse transcriptase and thereaction is primed by a population of random 9-mer oligonucleotides (SEQID NO:7). The product of the third DNA molecule synthesis reaction is apopulation of double-stranded nucleic acid molecules in which the firstRNA molecules are hybridized to the third DNA molecules. Thedouble-stranded nucleic acid hybrid is subjected to alkaline hydrolysisto hydrolyze the first RNA molecules. The third DNA molecules are thenpurified to remove nucleic acid molecules of less than about 100 bases.Purification is achieved by applying the third DNA molecule sample to acolumn that separates nucleic acid molecules on the basis of size.

The purified third DNA molecules are then labeled with one or more typesof dye. Any useful dye can be utilized, provided that the dye is linkedto the third DNA molecules by aminoally linkages.

Amplification Efficiency of the methods of the invention. FIG. 2 shows arepresentative embodiment of the methods of the invention, and alsoshows the amplification efficiency of each step of the representativeembodiment. Thus, the embodiment of the methods of the invention shownin FIG. 2 converts an amount of mRNA sufficient to conduct 0.5hybridization experiments, into an amount of complementary DNAsufficient to conduct 667 hybridization experiments. The hybridization,scanning and image analysis is conducted as described in Hughes et. al.,Nature Biotechnology 19:342-347 (2001). Hybridization experiments areconducted using oligonucleotide arrays consisting of 60-mers synthesizedas described by Hughes et. al., supra.

Reproducibility and accuracy of the methods of the invention. Todetermine if the methods of the invention yield reproducible results,independent amplifications of a single preparation of mRNA isolated fromJurkat cells were compared. Labeled products from each amplificationgave no false positives (P<0.01) when hybridized to a Human 25 kmicroarray. In addition, comparison of Jurkat and K562 samples amplifiedin duplicate revealed similar expression ratios when hybridized to aHuman 25 k array (r=0.99, P<0.01). Perfect duplication of geneexpression patterns would result in r=1.0.

In order to determine if tissure-specific gene expression patterns wereconserved through amplification, mRNA from the human Jurkat and K562cell lines was used to generate cDNA by a conventional, random-primed,reverse transcription method, as well as by the methods of the presentinvention. The expression ratios resulting from each method correlatedat r=0.95 (P<0.01). Perfect duplication of gene expression patternswould result in r=1.0, and profiles were said to be conserved if r>0.90.

Representation of full-length transcripts. To determine if the methodsof the invention generate cDNA representing full-length transcripts,amplification products, prepared in accordance with the methods of theinvention, were hybridized to microarrays containing 60mer probes tiledacross complete mRNA sequences for a number of human genes. Thedistribution of signal intensity consistently extended across wholetranscripts, independent of transcript size and distance from the 3′end; whereas a conventional oligo dT-primed reverse transcription methodresulted in a concentration of signal at the 3′ end. Due to this 3′ biasusing conventional oligo dT-primed reverse transcription, the signalintensity often did not extend beyond the untranslated region preventingthe detection of even the 3′-most coding sequences.

Nucleic acid samples of the invention. In another aspect, the presentinvention provides nucleic acid samples prepared in accordance with themethods of the invention. Thus, in one embodiment, the present inventionprovides DNA samples prepared by a method comprising the steps of: (a)utilizing an RNA template to enzymatically synthesize a first DNAmolecule that is complementary to at least 50 contiguous bases of RNAtemplate; (b) utilizing the first DNA molecule as a template toenzymatically synthesize a second DNA molecule thereby forming adouble-stranded DNA molecule wherein the first DNA molecule ishybridized to the second DNA molecule; (c) utilizing the first or secondDNA molecule of the double-stranded DNA molecule as a template toenzymatically synthesize a first RNA molecule that is complementary toeither the first DNA molecule or to the second DNA molecule; and (d)utilizing the first RNA molecule as a template to enzymaticallysynthesize a third DNA molecule that is complementary to the first RNAmolecule. The third DNA molecules can be linked to dye molecules byaminoallyl linkages.

In another embodiment, the present invention provides DNA samplesprepared by a method comprising the steps of: (a) utilizing an RNAtemplate to enzymatically synthesize a first DNA molecule that iscomplementary to at least 50 contiguous bases of the RNA template; (b)utilizing the first DNA molecule as a template to enzymaticallysynthesize a second DNA molecule thereby forming a double-stranded DNAmolecule wherein the first DNA molecule is hybridized to the second DNAmolecule; (c) enzymatically amplifying the double-stranded DNA molecule;(d) utilizing the first or second DNA molecule of the amplified,double-stranded, DNA molecule as a template to enzymatically synthesizea first RNA molecule that is complementary to either the first DNAmolecule or to the second DNA molecule; and (e) utilizing the first RNAmolecule as a template to enzymatically synthesize a third DNA moleculethat is complementary to the first RNA molecule. The third DNA moleculescan be linked to dye molecules by aminoallyl linkages.

The third DNA molecules produced by the subject methods finds use in avariety of applications. For example, third DNA molecules produced bythe methods of the invention may be labeled and employed to profile geneexpression in different populations of cells. In one embodiment, thirdDNA molecules are used for quantitative comparisons of gene expressionbetween different populations of cells or between populations of cellsexposed to different stimuli. For example, the third DNA molecules canbe used in expression profiling analysis on such platforms as DNAmicroarrays, for construction of “driver” for subtractive hybridizationassays, and the like. Especially facilitated by the subject methods arestudies of differential gene expression in mammalian cells or cellpopulations. The cells may be from blood (e.g., white cells, such as Tor B cells) or from tissue derived from solid organs, such as brain,spleen, bone, heart, vascular, lung, kidney, liver, pituitary, endocrineglands, lymph node, dispersed primary cells, tumor cells, or the like.

When used as probes to hybridize to a population of immobilized nucleicacid molecules, such as a population of nucleic acid moleculesimmobilized on a DNA array, DNA samples prepared in accordance with themethods of the invention exhibit a high level of hybridizationspecificity and sensitivity. For example, eight different transcriptswere synthesized and spiked into complex mRNA samples at differentratios. Genes were selected from the yeast genome based on similarity tothe physical characteristics of human genes (i.e., high GC content, ˜2.0kb length), and low potential for cross-hybridization with humansequences. The genes selected on this basis were YOR140W, YHR042W,YAL043C, YKR050W, YAL054C, YGL236C, YHL032C and YGL234W.

The coding sequences of each gene were PCR amplified from yeast genomicDNA and in vitro-transcribed to generate cRNA for spike-in experiments.These transcripts were added to mRNA from the Jurkat and K562 human celllines at different ratios between the two samples. The RNA mixtures werethen amplified and labeled according to the methods of the inventionbefore hybridization to an array with oligonucleotides representing eachtranscript. For each transcript, ten 60-mers were selected andsynthesized on a microarray as described by Hughes, et. al., supra. Themeasured expression ratios of oligonucleotides representing eachtranscript were averaged and plotted against the expected ratios.Accurate detection of spike-in ratios occurred at as low as 0.5 copiesper cell.

Methods for hybridizing a processed DNA sample to a population ofimmobilized nucleic acid molecules. In another aspect, the presentinvention provides methods for hybridizing a processed DNA sample to apopulation of immobilized nucleic acid molecules. The methods of thisaspect of the invention include the step of hybridizing a processed DNAsample to a population of immobilized nucleic acid molecules, whereinthe processed DNA sample is prepared in accordance with a method of thepresent invention.

Thus, in one embodiment, the present invention provides a method forhybridizing a processed DNA sample to a population of immobilizednucleic acid molecules, the method comprising the step of hybridizing aprocessed DNA sample to a population of immobilized nucleic acidmolecules, wherein the processed DNA sample is prepared by a methodcomprising the steps of: (a) utilizing an RNA template to enzymaticallysynthesize a first DNA molecule that is complementary to at least 50contiguous bases of the RNA template; (b) utilizing the first DNAmolecule as a template to enzymatically synthesize a second DNA moleculethereby forming a double-stranded DNA molecule wherein the first DNAmolecule is hybridized to the second DNA molecule; (c) utilizing thefirst or second DNA molecule of the double-stranded DNA molecule as atemplate to enzymatically synthesize a first RNA molecule that iscomplementary to either the first DNA molecule or to the second DNAmolecule; and (d) utilizing the first RNA molecule as a template toenzymatically synthesize a third DNA molecule that is complementary tothe first RNA molecule. The third DNA molecules can be linked to dyemolecules by aminoallyl linkages.

Conditions for hybridizing processed DNA samples of the invention toimmobilized nucleic acid molecules. Typically, hybridization conditionsare no more than 25° C. to 30° C. (for example, 110° C.) below themelting temperature (Tm) of the native duplex; see generally, Sambrooket al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Press, 1987; Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing, 1987).

Tm for nucleic acid molecules greater than about 100 bases can becalculated by the formula Tm=81.5+0.41%(G+C)— log(Na+). Foroligonucleotide molecules less than 100 bases in length, exemplaryhybridization conditions are 5 to 10° C. below Tm.

Preparation of microarrays. Nucleic acid molecules, that are to behybridized to a processed DNA sample in accordance with this aspect ofthe invention, can be immobilized by any art-recognized means. Forexample, nucleic acid molecules (such as DNA or RNA molecules) can beimmobilized to nitrocellulose, or to a synthetic membrane capable ofbinding nucleic acid molecules, or to a nucleic acid microarray, such asa DNA microarray. A DNA microarray, or chip, is a microscopic array ofDNA fragments, including synthetic oligonucleotides, disposed in adefined pattern on a solid support, wherein they are amenable toanalysis by standard hybridization methods (see, Schena, BioEssays18:427, 1996).

The DNA in a microarray may be derived, for example, from genomic orcDNA libraries, from fully sequenced clones, or from partially sequencedcDNAs known as expressed sequence tags (ESTs). Methods for obtainingsuch DNA molecules are generally known in the art (see, e.g., Ausubel etal., eds., 1994, Current Protocols in Molecular Biology, vol. 2, CurrentProtocols Publishing, New York). Again by way of example,oligonucleotides may be synthesized by conventional methods, such as themethods described herein.

Microarrays can be made in a number of ways, of which several aredescribed below. However produced, microarrays share certain preferredcharacteristics: The arrays are reproducible, allowing multiple copiesof a given array to be produced and easily compared with each other.Preferably the microarrays are small, usually smaller than 5 cm², andthey are made from materials that are stable under nucleic acidhybridization conditions. A given binding site or unique set of bindingsites in the microarray will specifically bind the product of a singlegene in the cell (or a nucleic acid molecule that represents the productof a single gene, such as a cDNA molecule that is complementary to all,or to part, of an mRNA molecule). Although there may be more than onephysical binding site (hereinafter “site”) per specific gene product,for the sake of clarity the discussion below will assume that there is asingle site.

In one embodiment, the microarray is an array of polynucleotide probes,the array comprising a support with at least one surface and at least100 different polynucleotide probes, each different polynucleotide probecomprising a different nucleotide sequence and being attached to thesurface of the support in a different location on the surface. Forexample, the nucleotide sequence of each of the different polynucleotideprobes can be in the range of 40 to 80 nucleotides in length. Forexample, the nucleotide sequence of each of the different polynucleotideprobes can be in the range of 50 to 70 nucleotides in length. Forexample, the nucleotide sequence of each of the different polynucleotideprobes can be in the range of 50 to 60 nucleotides in length.

In specific embodiments, the array comprises polynucleotide probes of atleast 2,000, 4,000, 10,000, 15,000, 20,000, 50,000, 80,000, or 100,000different nucleotide sequences.

In another embodiment, the nucleotide sequence of each polynucleotideprobe in the array is specific for a particular target polynucleotidesequence. In yet another embodiment, the target polynucleotide sequencescomprise expressed polynucleotide sequences of a cell or organism.

In a specific embodiment, the cell or organism is a mammalian cell ororganism. In another specific embodiment, the cell or organism is ahuman cell or organism.

In specific embodiments, the nucleotide sequences of the differentpolynucleotide probes of the array are specific for at least 50%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% of the genes in the genome of the cell or organism. Mostpreferably, the nucleotide sequences of the different polynucleotideprobes of the array are specific for all of the genes in the genome ofthe cell or organism.

In specific embodiments, the polynucleotide probes of the arrayhybridize specifically and distinguishably to at least 10,000, to atleast 20,000, to at least 50,000, to at least 80,000, or to at least100,000 different polynucleotide sequences.

In other specific embodiments, the polynucleotide probes of the arrayhybridize specifically and distinguishably to at least 90%, at least95%, or at least 99% of the genes or gene transcripts of the genome of acell or organism. Most preferably, the polynucleotide probes of thearray hybridize specifically and distinguishably to the genes or genetranscripts of the entire genome of a cell or organism.

In specific embodiments, the array has at least 100, at least 250, atleast 1,000, or at least 2,500 probes per 1 cm², preferably all or atleast 25% or 50% of which are different from each other.

In another embodiment, the array is a positionally addressable array (inthat the sequence of the polynucleotide probe at each position isknown).

In another embodiment, the nucleotide sequence of each polynucleotideprobe in the array is a DNA sequence. In another embodiment, the DNAsequence is a single-stranded DNA sequence. The DNA sequence may be,e.g., a cDNA sequence, or a synthetic sequence.

When a nucleic acid molecule that corresponds to an mRNA of a cell (suchas a third DNA molecule produced in the practice of the methods of theinvention for synthesizing a preparation of nucleic acid molecules) ismade and hybridized to a microarray under suitable hybridizationconditions, the level of hybridization to the site in the arraycorresponding to any particular gene will reflect the prevalence in thecell of mRNA transcribed from that gene. For example, when detectablylabeled (e.g., with a fluorophore) DNA complementary to the totalcellular mRNA is hybridized to a microarray, the site on the arraycorresponding to a gene (i.e., capable of specifically binding theproduct of the gene) that is not transcribed in the cell will havelittle or no signal (e.g., fluorescent signal), and a gene for which theencoded mRNA is prevalent will have a relatively strong signal.

In some embodiments, third DNA molecule populations prepared from RNAfrom two different cells are hybridized to the binding sites of themicroarray. In the case of drug responses one biological sample isexposed to a drug and another biological sample of the same type is notexposed to the drug. In the case of pathway responses, one cell isexposed to a pathway perturbation and another cell of the same type isnot exposed to the pathway perturbation. The third DNA molecules derivedfrom each of the two cell types are differently labeled so that they canbe distinguished. In one embodiment, for example, third DNA moleculesfrom a cell treated with a drug (or exposed to a pathway perturbation)is synthesized using a fluorescein-labeled NTP, and third DNA moleculesfrom a second cell, not drug-exposed, is synthesized using arhodamine-labeled NTP. When the two populations of third DNA moleculesare mixed and hybridized to the microarray, the relative intensity ofsignal from each population of third DNA molecules is determined foreach site on the array, and any relative difference in abundance of aparticular mRNA detected.

In the example described above, the third DNA molecule population fromthe drug-treated (or pathway perturbed) cell will fluoresce green whenthe fluorophore is stimulated and the third DNA molecule population fromthe untreated cell will fluoresce red. As a result, when the drugtreatment has no effect, either directly or indirectly, on the relativeabundance of a particular mRNA in a cell, the mRNA will be equallyprevalent in both cells and, upon synthesis of third DNA molecules inaccordance with the present invention, red-labeled and green-labeledthird DNA molecules will be equally prevalent. When hybridized to themicroarray, the binding site(s) for that species of RNA will emitwavelengths characteristic of both fluorophores (and appear brown incombination). In contrast, when the drug-exposed cell is treated with adrug that, directly or indirectly, increases the prevalence of the mRNAin the cell, the ratio of green to red fluorescence will increase. Whenthe drug decreases the mRNA prevalence, the ratio will decrease.

The use of a two-color fluorescence labeling and detection scheme todefine alterations in gene expression has been described, e.g., inSchena et al., 1995, Science 270:467-470, which is incorporated byreference in its entirety for all purposes. An advantage of using thirdDNA molecules labeled with two different fluorophores is that a directand internally controlled comparison of the mRNA levels corresponding toeach arrayed gene in two cell states can be made, and variations due tominor differences in experimental conditions (e.g., hybridizationconditions) will not affect subsequent analyses. However, it will berecognized that it is also possible to use third DNA molecules from asingle cell, and compare, for example, the absolute amount of aparticular mRNA in, e.g., a drug-treated or pathway-perturbed cell andan untreated cell.

Preparation of nucleic acid molecules for immobilization on microarrays.As noted above, the “binding site” to which a particular, cognate,nucleic acid molecule specifically hybridizes is usually a nucleic acidor nucleic acid analogue attached at that binding site. In oneembodiment, the binding sites of the microarray are DNA polynucleotidescorresponding to at least a portion of each gene in an organism'sgenome. These DNAs can be obtained by, for example, polymerase chainreaction (PCR) amplification of gene segments from genomic DNA, cDNA(e.g., by reverse transcription or RT-PCR), or cloned sequences. Nucleicacid amplification primers are chosen, based on the known sequence ofthe genes or cDNA, that result in amplification of unique fragments(i.e., fragments that do not share more than 10 bases of contiguousidentical sequence with any other fragment on the microarray). Computerprograms are useful in the design of primers with the requiredspecificity and optimal amplification properties. See, e.g., Oligoversion 5.0 (National Biosciences). In the case of binding sitescorresponding to very long genes, it will sometimes be desirable toamplify segments near the 3′ end of the gene so that when oligo-dTprimed DNA probes are hybridized to the microarray, less-than-fulllength probes will bind efficiently. Typically each gene fragment on themicroarray will be between about 50 bp and about 2000 bp, more typicallybetween about 100 bp and about 1000 bp, and usually between about 300 bpand about 800 bp in length.

Nucleic acid amplification methods are well known and are described, forexample, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methodsand Applications, Academic Press Inc., San Diego, Calif., which isincorporated by reference in its entirety for all purposes. It will beapparent that computer controlled robotic systems are useful forisolating and amplifying nucleic acids.

An alternative means for generating the nucleic acid for the microarrayis by synthesis of synthetic polynucleotides or oligonucleotides, e.g.,using N-phosphonate or phosphoramidite chemistries (e.g., Froehler etal., 1986, Nucleic Acid Res 14:5399-5407). Synthetic sequences aretypically between about 15 and about 100 bases in length, such asbetween about 20 and about 50 bases.

In some embodiments, synthetic nucleic acids include non-natural bases,e.g., inosine. Where the particular base in a given sequence is unknownor is polymorphic, a universal base, such as inosine or 5-nitroindole,may be substituted. Additionally, it is possible to vary the charge onthe phosphate backbone of the oligonucleotide, for example, bythiolation or methylation, or even to use a peptide rather than aphosphate backbone. The making of such modifications is within the skillof one trained in the art.

As noted above, nucleic acid analogues may be used as binding sites forhybridization. An example of a suitable nucleic acid analogue is peptidenucleic acid (see, e.g., Egholm et al., 1993, Nature 365:566-568; seealso U.S. Pat. No. 5,539,083).

In another embodiment, the binding (hybridization) sites are made fromplasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags),or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209). In yetanother embodiment, the polynucleotide of the binding sites is RNA.

Attaching nucleic acids to the solid support. The nucleic acid oranalogue are attached to a solid support, which may be made from glass,silicon, plastic (e.g., polypropylene, nylon, polyester),polyacrylamide, nitrocellulose, cellulose acetate or other materials. Ingeneral, non-porous supports, and glass in particular, are preferred.The solid support may also be treated in such a way as to enhancebinding of oligonucleotides thereto, or to reduce non-specific bindingof unwanted substances thereto. Preferably, the glass support is treatedwith polylysine or silane to facilitate attachment of oligonucleotidesto the slide.

Methods of immobilizing DNA on the solid support may include directtouch, micropipetting (see, e.g., Yershov et al., Proc. Natl. Acad. Sci.USA 93(10):4913-4918, 1996), or the use of controlled electric fields todirect a given oligonucleotide to a specific spot in the array (see,e.g., U.S. Pat. No. 5,605,662). DNA is typically immobilized at adensity of 100 to 10,000 oligonucleotides per cm² such as at a densityof about 1000 oligonucleotides per cm².

A preferred method for attaching the nucleic acids to a surface is byprinting on glass plates, as is described generally by Schena et al.,1995, Science 270:467-470. This method is especially useful forpreparing microarrays of cDNA. (See also DeRisi et al., 1996, NatureGenetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; andSchena et al., Proc. Natl. Acad. Sci. USA, 93(20):10614-19, 1996.)

In an alternative to immobilizing pre-fabricated oligonucleotides onto asolid support, it is possible to synthesize oligonucleotides directly onthe support (see, e.g., Maskos et al., Nucl. Acids Res. 21:2269-70,1993; Fodor et al., Science 251:767-73, 1991; Lipshutz et al., 1999,Nat. Genet. 21(1 Suppl):20-4). Methods of synthesizing oligonucleotidesdirectly on a solid support include photolithography (see Fodor et al.,Science 251:767-73, 1991; McGall et al., Proc. Natl. Acad. Sci. (USA)93:13555-60, 1996) and piezoelectric printing (Lipshutz et al., 1999,Nat. Genet. 21(1 Suppl):20-4).

In one embodiment, a high-density oligonucleotide array is employed.Techniques are known for producing arrays containing thousands ofoligonucleotides complementary to defined sequences, at definedlocations on a surface using photolithographic techniques for synthesisin situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al.,1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al., 1996,Nature Biotechnol. 14:1675-80; U.S. Pat. No. 5,578,832; U.S. Pat. No.5,556,752; and U.S. Pat. No. 5,510,270; each of which is incorporated byreference in its entirety for all purposes) or other methods for rapidsynthesis and deposition of defined oligonucleotides (Lipshutz et al.,1999, Nat. Genet. 21(1 Suppl):20-4.).

In one embodiment, microarrays are manufactured by means of an ink jetprinting device for oligonucleotide synthesis, e.g., using the methodsand systems described by Blanchard in International Patent PublicationNo. WO 98/41531, published Sep. 24, 1998; Blanchard et al., 1996,Biosensors and Bioeletronics 11:687-690; Blanchard, 1998, in SyntheticDNA Arrays in Genetic Engineering, Vol. 20, J. K. Setlow, Ed., PlenumPress, New York at pages 111-123; U.S. Pat. No. 6,028,189 to Blanchard.Specifically, the oligonucleotide probes in such microarrays arepreferably synthesized in arrays, e.g., on a glass slide, by seriallydepositing individual nucleotide bases in “microdroplets” of a highsurface tension solvent such as propylene carbonate. The microdropletshave small volumes (e.g., 100 pL or less, more preferably 50 pL or less)and are separated from each other on the microarray (e.g., byhydrophobic domains) to form circular surface tension wells which definethe locations of the array elements (i.e., the different probes).

Other methods for making microarrays, e.g., by masking (Maskos andSouthern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used. Inprincipal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.), could be used, although, as will berecognized by those of skill in the art, very small arrays will bepreferred because hybridization volumes will be smaller.

Signal detection and data analysis. When fluorescently labeled probesare used, the fluorescence emissions at each site of a transcript arraycan be detected by scanning confocal laser microscopy. In oneembodiment, a separate scan, using the appropriate excitation line, iscarried out for each of the two fluorophores used. Alternatively, alaser can be used that allows simultaneous specimen illumination atwavelengths specific to the two fluorophores and emissions from the twofluorophores can be analyzed simultaneously (see Shalon et al., 1996,Genome Research 6:639-645, which is incorporated by reference in itsentirety for all purposes). One embodiment, the arrays are scanned witha laser fluorescent scanner with a computer controlled X-Y stage and amicroscope objective. Sequential excitation of the two fluorophores isachieved with a multi-line, mixed gas laser and the emitted light issplit by wavelength and detected with two photomultiplier tubes.Fluorescence laser scanning devices are described in Shalon et al.,1996, Genome Res. 6:639-645 and in other references cited herein.Alternatively, the fiber-optic bundle described by Ferguson et al.,1996, Nature Biotechnol. 14:1681-1684, may be used to monitor mRNAabundance levels at a large number of sites simultaneously.

Signals are recorded and, in one embodiment, analyzed by computer, e.g.,using a 12 bit analog to digital board. In one embodiment the scannedimage is despeckled using a graphics program (e.g., Hijaak GraphicsSuite) and then analyzed using an image gridding program that creates aspreadsheet of the average hybridization at each wavelength at eachsite. If necessary, an experimentally determined correction for “crosstalk” (or overlap) between the channels for the two fluors may be made.For any particular hybridization site on the transcript array, a ratioof the emission of the two fluorophores can be calculated. The ratio isindependent of the absolute expression level of the cognate gene, but isuseful for genes whose expression is significantly modulated by drugadministration, gene deletion, or any other tested event.

The relative abundance of an mRNA in two biological samples is scored asa perturbation and its magnitude determined (i.e., the abundance isdifferent in the two sources of mRNA tested), or as not perturbed (i.e.,the relative abundance is the same). In various embodiments, adifference between the two sources of RNA of at least a factor of about25% (RNA from one source is 25% more abundant in one source than theother source), more usually about 50%, even more often by a factor ofabout 2 (twice as abundant), 3 (three times as abundant) or 5 (fivetimes as abundant) is scored as a perturbation.

Preferably, in addition to identifying a perturbation as positive ornegative, it is advantageous to determine the magnitude of theperturbation. This can be carried out, as noted above, by calculatingthe ratio of the emission of the two fluorophores used for differentiallabeling, or by analogous methods that will be readily apparent to thoseof skill in the art.

By way of example, two samples, each labeled with a different fluor, arehybridized simultaneously to permit differential expressionmeasurements. If neither sample hybridizes to a given spot in the array,no fluorescence will be seen. If only one hybridizes to a given spot,the color of the resulting fluorescence will correspond to that of thefluor used to label the hybridizing sample (for example, green if thesample was labeled with Cy3, or red, if the sample was labeled withCy5). If both samples hybridize to the same spot, an intermediate coloris produced (for example, yellow if the samples were labeled withfluorescein and rhodamine). Then, applying methods of patternrecognition and data analysis known in the art, it is possible toquantify differences in gene expression between the samples. Methods ofpattern recognition and data analysis are described in e.g.,International Publication WO 00/24936, dated May 4, 2000, which isincorporated by reference herein.

The following examples merely illustrate the best mode now contemplatedfor practicing the invention, but should not be construed to limit theinvention.

EXAMPLE 1 cDNA Synthesis and RNA Amplification for the Preparation ofCy3— and Cy5-Labeled RNA Targets for Gene Expression Monitoring

This Example describes the synthesis of a population of third DNAmolecules prepared in accordance with the methods of the invention.

Isolation of total RNA. Total RNA was isolated from Jurkat and K562 celllines by gentle lysis with the RNeasy Purification Kit (QIAGEN Inc.,Valencia, Calif.).

Isolation of messenger RNA. Poly A⁺ RNA was isolated from the total RNAto provide the template mRNA used in the experiment. Poly A⁺ RNA wasisolated by two sequential chromatographic purifications over oligo dTcellulose (New England Biolabs, Beverly, Mass.) using establishedprotocols as described by Ausubel, F. M. et al. in Current Protocols inMolecular Biology 13.12.1-13.12.5 (eds. Ausubel, F. M. et al.) (JohnWiley & Sons, New York, 1993). 1.5 μg of cytoplasmic mRNA was used inthe subsequent procedure.

Synthesis of first DNA molecules. The following reverse transcriptionreaction was performed. The first primer mixture, called ShDNP256,included a multiplicit of primer molecules that each had the followingnucleic acid sequence:

-   -   ShDNP256 5′ TAG ATG CTG TTG NNN NNN NNN 3′ (SEQ ID NO:3),        wherein N represents any nucleic acid residue (e.g., A, C, G, T        or I).

For each sample of template mRNA, the mRNA sample and the first primermixture were added to an eppendorf tube in the following amounts:

-   -   mRNA (1.5 μg) 13.4 μl (adjust volume with water)    -   100 μM ShDNP256 (SEQ ID NO:3)1.6 μl    -   15.0 μl

The preparation was incubated for 10 minutes (min) at 70° C., then for 5min on ice, and then for 10 min at room temperature. The followingreagents were prepared as a pre-mix, and a 2511 aliquot was added toeach sample (containing mRNA and first primer mixture) and mixed well:10 mM dNTPs 2.0 μl 5X RT buffer 8.0 μl 50 mM MgCl₂ 4.0 μl 100 mM DTT 4.0μl water 6.0 μl Superscript II (200 U/μl) 1.0 μl 25.0 μl 

The reverse transcriptase utilized to synthesize the first DNA moleculeswas SUPERSCRIPT II RNase H—Reverse Transcriptase (item no. 18064-014,Gibco-BRL, Rockville, Md.). The composition of 5×RT buffer (suppliedwith the SUPERSCRIPT II enzyme) was 250 mM Tris-HCl (pH 8.3), 375 mMKCl, 15 mM MgCl₂ (Gibco-BRL, Rockville, Md.). The water added to thefirst DNA molecule synthesis mixture was DNase/RNase-free water (itemno. 10977-015, Gibco-BRL, Rockville, Md.). Samples were incubated for 20min at 42° C.

Alkaline hydrolysis of template RNA and purification of first DNAmolecules. 20 μl of NaOH/EDTA (1:1 mix of 1 N NaOH and 0.5 M EDTA) wasthen added to each sample containing the products of the foregoingreverse transcription reaction. Samples were incubated for 20 min at 65°C. 20 μl of 1 M Tris-HCl, pH 7.6 was added and mixed. 20 μl of water wasadded.

The first DNA molecules in the treated samples were purified withQIAquick spin columns. 50 μl of Buffer PB (5:1 buffer to reactionvolume) was added to each sample and mixed. Buffer PB was supplied withthe QIAquick PCR Purification Kit (item no. 28106, QIAGEN Inc.,Valencia, Calif.). Each sample was then applied to a column, spun for 1min at top speed in a microcentrifuge, re-applied to the column and spunagain. The flow-through from the column was discarded. The sample wasthen washed with 500 μl of Buffer PE, spun in the microcentrifuge, andthe flow-through discarded. This wash was then repeated a second time.The sample was spun for an additional 1 min to remove residual PE.Buffer PE was supplied with the QIAquick PCR Purification Kit (item no.28106, QIAGEN Inc., Valencia, Calif.). The bound sample was eluted fromthe column by adding 50 μl of Buffer EB (10 mM Tris-HCl, pH 8.5), andincubating at room temp for 1 minute before spinning. The elution stepwas repeated, then the entire 100 μl eluate was quantitated in a 96-wellUV Plate (Costar item no. 3635, Corning Inc., Corning, N.Y.) with theSPECTRAmax PLUS 384 Microplate Reader (item no. 0200-3855, MolecularDevices Corp., Sunnyvale, Calif.). The procedure typically yielded450-600 ng of first DNA molecules per reverse transcription reaction.Each sample was concentrated to 18 μl with a Microcon-30 (Millipore) orspeed-vac apparatus.

Synthesis of second DNA molecules. the first DNA molecules were used astemplates to synthesize complementary second DNA molecules as follows.Second DNA molecule synthesis was primed using a second primer mixturecalled ShT7N9. Each primer of the second primer mixture had thefollowing nucleic acid sequence:

-   -   ShT7N9 5′ ACTA TAG GGA GAN NNN NNN NN 3′ (SEQ ID NO:2)

Each second DNA molecule synthesis reaction included the followingcomponents mixed in an eppendorf tube: First DNA molecules 18.0 μl 100μM ShT7N9(SEQ ID NO: 2)  2.0 μl 20.0 μl

Each reaction mixture was incubated for 5 min at 70° C., then incubatedfor 10 min at room temperature. The following reagents were then addedto each reaction mixture and mixed well: RNase free water 21.5 μl React2 buffer  5.0 μl 10 M dNTPs  2.5 μl Klenow (5 U/μl)  1.0 μl 30.0 μl

The water added to the second DNA molecule synthesis mixture wasDNase/RNase-free water (item no. 10977-015, Gibco-BRL, Rockville, Md.).The Klenow fragment of DNA Polymerase I was obtained from Gibco-BRL,Rockville, Md. (item no. 18012-039). The composition of 10× React 2Buffer was 500 mM Tris-HCl (pH 8.0), 100 mM MgCl₂, 500 mM NaCl(Gibco-BRL, Rockville, Md.). Each reaction mixture was incubated for 1hour at 37° C., then incubated for 2 min at 65° C.

Purification of double-stranded DNA molecules. The double-stranded DNAreaction products were purified with QIAquick spin columns (QIAGEN,Inc., Valencia, Calif.). 50 μl of water was added to each reactionmixture and the QIAquick spin column purification steps carried out asdescribed above for the purification of the first DNA molecules.

Amplification of double-stranded DNA molecules. Using spectrophotometricquantitation of cDNA, 150 ng of each second strand synthesis product wasadded to a PCR tube and adjusted to 25 μl with DNase/RNase-free water. Apre-mix of the following reagents was made, and for each double-strandedDNA sample, 75 μl of the pre-mix was aliquoted into a PCR tube and mixedwell. The T7 and DP256 primers had the following sequences: (SEQ IDNO:5) T7 5′ AAT TAA TAC GAC TCA CTA TAG GGA GA 3′ (SEQ ID NO:6) DP256 5′GTT CGA GAC CTC TAG ATG CTG TTG 3′

RNase free water 48.0 μl 10x PCR buffer 10.0 μl 50 mM MgCl₂  3.0 μl 1 mMdNTPs 10.0 μl 100 μM T7 primer (SEQ ID NO: 5)  1.0 μl 100 μM DP256primer(SEQ ID NO: 6)  1.0 μl Taq polymerase (5 U/μl)  2.0 μl 75.0 μl

The water added to the amplification mixture was DNase/RNase-free water(item no. 10977-015, Gibco-BRL, Rockville, Md.). The composition of10×PCR Buffer was 200 mM Tris-HCl (pH 8.4), 500 mM KCl (Gibco-BRL,Rockville, Md.). The 1 mM dNTPs were diluted from a 10 mM dNTP mixpurchased from Gibco-BRL, Rockville, Md. (item no. 18427-013). The T7primer and DP256 primer were purchased from New England Biolabs,Beverly, Mass. The Taq DNA Polymerase was purchased from Gibco-BRL,Rockville, Md. (item no. 18038-042). The thermal cycler was started inadvance and sample tubes were added when the temperature reached 94° C.The PCR reaction was run under the following cycle: 1 cycle of: 94° C.for 5 min 2 cycles of: 94° C. for 45 sec 40° C. for 2 min 72° C. for 4min 8 cycles of: 94° C. for 45 sec 55° C. for 2 min 72° C. for 4 min

The reaction product was purified with a QIAquick spin column asdescribed above for the purification of the first DNA molecules. All thePCR reactions derived from the same mRNA template sample were pooled.100 μl of eluate was quantitated in a 96-well UV Plate as describedabove. The typical yield was 1.2-1.8 μg of double-stranded DNA per PCRreaction.

Synthesis of the first RNA molecules. For each sample, 500 ng of PCRproduct was aliquoted into an eppendorf tube and the volume was adjustedto 40 μl with DNase/RNase-free water. A pre-mix of the followingreagents was made, and 40 μl was aliquoted into each sample and mixedwell: water  4.8 μl 5X Transcription buffer 16.00 μl  100 mM DTT 6.00 μl25 mM NTPs 8.00 μl 200 mM MgCl₂ 3.30 μl RNAGuard (36 U/μl) 0.50 μlInorganic Pyrophosphate (2000 U/ml)  0.6 μl T7 RNA Polymerase (2.5kU/μl)  0.8 μl 40.00 μl 

The water added to the RNA synthesis mixture was DNase/RNase-free water(item no. 10977-015, Gibco-BRL, Rockville, Md.). The composition of 5×Transcription Buffer was 200 mM Tris-HCl (pH 7.5), 50 mM NaCl, 30 mMMgCl₂, 10 mM spermidine (item no. BP1001, EPICENTRE Technologies,Madison, Wis.). The 25 mM NTP's were diluted from a 100 mM NTP Set (itemno. 27-2025-01, Pharmacia Biotech, Piscataway, N.J.). RNAguardRibonuclease Inhibitor was purchased from Pharmacia Biotech, Piscataway,N.J. (item no. 27-0815-01). Inorganic Pyrophosphatase was purchased fromNew England Biolabs, Beverly, Mass. (item no. M0296S). The samples wereincubated for 16 hours at 42° C. The samples were then incubated for 5min at 70° C.

Purification of the first RNA molecules. The samples were purified withthe RNeasy Purification Kit (QIAGEN Inc., Valencia, Calif.) as follows:

-   -   1. Add 20 μl of RNase-free water to the reaction mixture.    -   2. Add 350 μl of RLT Buffer and mix. The RLT Buffer was supplied        with the RNeasy Purification Kit.    -   3. Add 250 μl of EtOH and mix.    -   4. Apply to spin column and spin for 20 sec at 14,000 rpm.    -   5. Reload sample onto same spin column, spin and discard        flow-through.    -   6. Apply 500 μl of 80% EtOH, spin and discard flow-through.    -   7. Repeat wash step.    -   8. After discarding the flow-through, spin for 1 min to remove        residual EtOH.    -   9. Elute by adding 50 μl of 70° C. water and incubate for 1 min        at room temperature before spinning.    -   10. Repeat the elution step.

5 μl of eluate was added to 95 μl of TE (10 mM Tris-HCl, pH 7.3, 0.1 mMEDTA) and quantitated in a 96-well UV Plate as described above. Thetypical yield was 85-100 μg of first RNA molecules per RNA polymerasereaction.

Synthesis of third DNA molecules. First RNA molecules were used astemplates for the synthesis of complementary third DNA molecules. Thestarting material [for one sample] was 3.0 μg of first RNA moleculespurified from the preceding RNA polymerase reaction. For each sample,the following reagents were added to an eppendorf tube (the volume ofRNA was adjusted in Dnase/Rnase-free water): RNA (3.0 μg) 11.0 μl N9primer (SEQ ID NO: 7) (1 μg/μl)  4.0 μl 15.0 μl

The mixture was incubated for 10 min at 70° C., for 5 min on ice, thenfor 10 min at room temperature. A pre-mix of the following reagents wasmade and a 35 μl aliquot was added to each sample (containing N9 primer(SEQ ID NO:7) hybridized to first RNA molecules) and mixed well: 10 mMdNTPs 2.5 μl 10 mM aa-dUTP 2.5 μl 5X RT buffer 10.0 μl  50 mM MgCl₂ 5.0μl 100 mM DTT 5.0 μl DNase/RNase-free water 7.5 μl Superscript II (200U/μl) 2.5 μl 35.0 μl 

10 mM aa-dUTP was purchased from Sigma, St. Louis, Mo. (item no. A0410). The composition of 5×RT buffer was 250 mM Tris-HCl (pH 8.3), 375mM KCl, 15 mM MgCl₂ (Gibco-BRL, Rockville, Md.). The samples wereincubated at 42° C. for 20 min.

Alkaline hydrolysis of first RNA molecules and purification of third DNAmolecules. 25 μl of NaOH/EDTA (1:1 mix of 1N NaOH and 0.5M EDTA) wasthen added. The samples were incubated at 65° C. for 20 min, then 25 μlof 1M Tris-HCl, pH 7.6 was added and mixed. Each sample was purifiedwith a QIAquick (QIAGEN) spin column as follows:

-   -   1. Add 500 μl of Buffer PB (5:1 buffer to reaction volume) and        mix.    -   2. Apply to column.    -   3. Spin for 1 min. at top speed in a microfuge.    -   4. Re-apply and spin again.    -   5. Discard flow-through.    -   6. Wash with 500 μl of Buffer PE, spin and discard flow-through.    -   7. Repeat wash one time.    -   8. Spin for an additional 1 min. to remove residual PE    -   9. Elute by adding 50 μl of Buffer EB (10 mM Tris-HCl, pH 8.5)        and incubating at room temp for 1 min. before spinning    -   10. Repeat elution step

The entire 100 μl eluate from each sample was quantitated in a 96-wellplate. The typical yield was 1.5-2.1 μg of third DNA molecules perreverse transcription reaction. Optionally, each sample can beconcentrated to approximately 10 μl in a Microcon-30 (Millipore). Eachsample was dried down in a speed-vac and resuspended in 3.5 μl of water.

Labelling the third DNA molecules with Cy dye. Each cDNA sample wasresusupended in 8 μl of 1× bicarbonate buffer (item no. C-3041, Sigma,St. Louis, Mo.). Lyophilized Cy3-NHS-ester (item no. Q13108, PharmaciaBiotech, Piscataway, N.J.) and Cy5-NHS-ester dyes (item no. Q13108,Pharmacia Biotech, Piscataway, N.J.) were resuspended in 367 and 400 μlof dimethyl sulfoxide (item no. D-8779, Sigma, St. Louis, Mo.),respectively. Then 8 μl of Cy3 or Cy5 dye was added to each sample andmixed thoroughly. Samples were then incubated at room temperature for 1hour in the dark. Reactions were stopped by adding 8 μl of 4Mhydroxylamine (item no. H-2391, Sigma, St. Louis, Mo.) followed by a 10minute incubation in the dark at room temperature. Unincorporated dyemolecules were removed with the QIAquick PCR Purification Kit (item no.28106, QIAGEN Inc., Valencia, Calif.) as described earlier. The percentdye incorporation and cDNA yield were determined spectrophotometrically.Pairs of Cy3/Cy5-labelled cDNA samples were combined and hybridized toDNA microarrays.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of synthesizing a preparation of nucleic acid molecules, themethod comprising the steps of: (a) utilizing an RNA template toenzymatically synthesize a first DNA molecule that is complementary toat least 50 contiguous bases of said RNA template; (b) utilizing thefirst DNA molecule as a template to enzymatically synthesize a secondDNA molecule, thereby forming a double-stranded DNA molecule wherein thefirst DNA molecule is hybridized to the second DNA molecule; (c)utilizing the first or second DNA molecule of the double-stranded DNAmolecule as a template to enzymatically synthesize a first RNA moleculethat is complementary to either the first DNA molecule or to the secondDNA molecule; and (d) utilizing the first RNA molecule as a template toenzymatically synthesize a third DNA molecule that is complementary tothe first RNA molecule.
 2. The method of claim 2 wherein thedouble-stranded DNA molecule is enzymatically amplified before utilizingthe first or second DNA molecule of the double-stranded DNA molecule asa template to enzymatically synthesize a first RNA molecule.
 3. Themethod of claim 2, wherein the RNA template is messenger RNA.
 4. Themethod of claim 2, wherein the first DNA molecule is synthesized usingreverse transcriptase.
 5. The method of claim 2, wherein the synthesisof the first DNA molecule is primed using a first primer mixturecomprising a multiplicity of first primers, wherein each of the firstprimers comprises a random sequence portion, and a defined sequenceportion.
 6. The method of claim 5, wherein the defined sequence portionis located 5′ to the random sequence portion.
 7. The method of claim 6,wherein the defined sequence portion comprises the nucleic acid sequenceof an RNA polymerase promoter.
 8. The method of claim 7, wherein thedefined sequence portion comprises the nucleic acid sequence set forthin SEQ ID NO:1.
 9. The method of claim 6, wherein each of the firstprimers consists of the nucleic acid sequence of primer ShT7N9 set forthin SEQ ID NO.
 2. 10. The method of claim 5, wherein the random sequenceportion consists of from 4 to 20 nucleic acid residues.
 11. The methodof claim 5, wherein the random sequence portion consists of from 4 to 15nucleic acid residues.
 12. The method of claim 5, wherein the randomsequence portion consists of from 6 to 9 nucleic acid residues.
 13. Themethod of claim 5, wherein the random sequence portion consists of 9nucleic acid residues.
 14. The method of claim 5 wherein each of thefirst primers consists of the nucleic acid sequence of primer ShDNP256set forth in SEQ ID NO:3.
 15. The method of claim 5, further comprisingutilizing a poly-dT primer comprising a poly-dT portion and a definedsequence portion, wherein the poly-dT portion is located 5′ to thedefined sequence portion.
 16. The method of claim 15, wherein thesequence of the defined sequence portion of the poly-dT primer isidentical to the sequence of the defined sequence portion of the primersof the first primer mixture.
 17. The method of claim 15, wherein thepoly-dT portion of the poly-dT primer consists of from 5 to 25 nucleicacid residues.
 18. The method of claim 15, wherein the poly-dT portionof the poly-dT primer consists of from 15 to 25 nucleic acid residues.19. The method of claim 15, wherein the poly-dT portion of the poly-dTprimer consists of 18 nucleic acid residues.
 20. The method of claim 5,further comprising the step of hydrolyzing the RNA template, andsubstantially removing the first primer mixture, after synthesizing thefirst DNA molecule and before synthesizing the second DNA molecule. 21.The method of claim 2, wherein the second DNA molecule is synthesizedusing the Klenow fragment of DNA polymerase I.
 22. The method of claim5, wherein the synthesis of the second DNA molecule is primed using asecond primer mixture comprising a multiplicity of second primermolecules, wherein each second primer molecule comprises a randomsequence portion and a defined sequence portion, wherein the sequence ofthe defined sequence portion of the second primer molecules is differentfrom the sequence of the defined sequence portion of the first primermolecules.
 23. The method of claim 22, wherein the defined sequenceportion of the second primer molecules is located 5′ to the randomsequence portion of the second primer molecules.
 24. The method of claim22, wherein the defined sequence portion of each second primer moleculecomprises the nucleic acid sequence of an RNA polymerase promoter. 25.The method of claim 24 wherein the defined sequence portion of eachfirst primer molecule does not comprise the nucleic acid sequence of anRNA polymerase promoter.
 26. The method of claim 24, wherein the definedsequence portion of each second primer molecule comprises the nucleicacid sequence set forth in SEQ ID NO.
 1. 27. The method of claim 24,wherein each second primer molecule consists of the nucleic acidsequence of primer ShT7N9 set forth in SEQ ID NO.
 2. 28. The method ofclaim 22 wherein the defined sequence portion of each first primermolecule comprises the nucleic acid sequence of an RNA polymerasepromoter, and the defined sequence portion of each second primermolecule does not comprise the nucleic acid sequence of an RNApolymerase promoter.
 29. The method of claim 22 wherein the secondprimer molecule consists of the nucleic acid sequence of primerShDNP256, set forth in SEQ ID NO:3.
 30. The method of claim 22 whereineach first primer consists of the nucleic acid sequence of primerShDNP256 set forth in SEQ ID NO:3, and each second primer consists ofthe nucleic acid sequence of primer ShT7N9, set forth in SEQ ID NO:2.31. The method of claim 2, wherein the double-stranded DNA molecule isamplified using a polymerase chain reaction comprising from 1 to 25amplification cycles.
 32. The method of claim 31, wherein the number ofamplification cycles is from 5 to
 15. 33. The method of claim 31,wherein the number of amplification cycles is
 10. 34. The method ofclaim 2, further comprising the step of purifying the amplified,double-stranded, DNA molecule before synthesizing the first RNAmolecule.
 35. The method of claim 2, wherein the first DNA molecule ofthe amplified, double-stranded, DNA molecule is utilized as a templateto enzymatically synthesize the first RNA molecule.
 36. The method ofclaim 35, wherein the first DNA molecule of the amplified,double-stranded, DNA molecule comprises a T7 RNA polymerase promoterthat promotes synthesis of the first RNA molecule.
 37. The method ofclaim 36, wherein the T7 RNA polymerase promoter comprises the nucleicacid sequence set forth in SEQ ID NO.
 1. 38. The method of claim 36,wherein the T7 RNA polymerase promoter consists of the nucleic acidsequence set forth in SEQ ID NO.
 1. 39. The method of claim 2, whereinthe second DNA molecule of the amplified, double-stranded, DNA moleculeis utilized as a template to synthesize the first RNA molecule.
 40. Themethod of claim 39, wherein the second DNA molecule comprises a T7 RNApolymerase promoter which promotes the synthesis of the first RNAmolecule.
 41. The method of claim 40, wherein the T7 RNA polymerasepromoter comprises the nucleic acid sequence set forth in SEQ ID NO. 1.42. The method of claim 40, wherein the T7 RNA polymerase promoterconsists of the nucleic acid sequence set forth in SEQ ID NO.
 1. 43. Themethod of claim 2, further comprising the step of purifying the firstRNA molecule before synthesizing the third DNA molecule, wherein thepurification step removes substantially all nucleic acid molecules lessthan 100 bases long.
 44. The method of claim 2 wherein the third DNAmolecule is synthesized using reverse transcriptase, and the synthesisof the third DNA molecule is primed using a population of randomprimers.
 45. The method of claim 44 wherein at least 99 percent of therandom primers consist of nine nucleotides.
 46. The method of claim 2wherein the third DNA molecule is purified to remove substantially allnucleic acid molecules less than 100 bases long.
 47. The method of claim2 wherein dye molecules are joined to the third DNA molecule byaminoallyl linkages.
 48. The method of claim 47 wherein the dye is a Cydye.
 49. A method of synthesizing a preparation of nucleic acidmolecules, the method comprising the steps of: (a) utilizing an RNAtemplate to enzymatically synthesize a first DNA molecule that iscomplementary to at least 50 contiguous bases of the RNA template,wherein: (i) the first DNA molecule is synthesized using reversetranscriptase; (ii) the synthesis of the first DNA molecule is primedusing a first primer mixture comprising a multiplicity of first primers,wherein each of the first primers comprises a random sequence portionand a defined sequence portion located 5′ to the random sequenceportion, wherein the defined sequence portion comprises a nucleic acidsequence selected from the group consisting of SEQ ID NO:2 and SEQ IDNO:3; (b) hydrolyzing the template RNA and removing substantially all ofthe first primer mixture after synthesis of the first DNA molecule; (c)utilizing the first DNA molecule as a template to enzymaticallysynthesize a second DNA molecule, thereby forming a double-stranded DNAmolecule wherein the first DNA molecule is hybridized to the second DNAmolecule, wherein: (i) the second DNA molecule is synthesized using theKlenow fragment of DNA polymerase I; (ii) the synthesis of the secondDNA molecule is primed using a second primer mixture comprising amultiplicity of second primer molecules, wherein each second primermolecule comprises a random sequence portion and a defined sequenceportion, wherein the sequence of the defined sequence portion of eachsecond primer molecule is different from the sequence of the definedsequence portion of each first primer molecule, and wherein the secondprimer defined sequence portion comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3; (d)removing substantially all of the second primer mixture after synthesisof the second DNA molecule; (e) amplifying the double-stranded DNAmolecule using a polymerase chain reaction comprising from 5 to 15amplification cycles, wherein the polymerase chain reaction is primedwith a first PCR primer population and a second PCR primer population,wherein the first PCR primer population consists essentially of primermolecules consisting of the sequence set forth in SEQ ID NO:5, and thesecond PCR primer population consists essentially of primer moleculesconsisting of the sequence set forth in SEQ ID NO:6; (f) removingsubstantially all of the PCR primer mixture after amplification of thedouble-stranded DNA molecule; (g) utilizing the first or second DNAmolecule of the amplified, double-stranded, DNA molecule as a templateto synthesize, using an RNA polymerase molecule, a first RNA moleculethat is complementary to either the first DNA molecule or to the secondDNA molecule; (h) purifying the first RNA molecule to removesubstantially all nucleic acid molecules less than 100 bases long; (i)utilizing the first RNA molecule as a template to enzymaticallysynthesize a third DNA molecule that is complementary to the first RNAmolecule, wherein the third DNA molecule is synthesized using reversetranscriptase and the synthesis of the third DNA molecule is primedusing a population of random primers wherein substantially all of therandom primers consist of 9 bases; and (j) joining Cy dye molecules tothe third DNA molecule by aminoallyl linkages.
 50. A DNA sample preparedby a method comprising the steps of: (a) utilizing an RNA template toenzymatically synthesize a first DNA molecule that is complementary toat least 50 contiguous bases of said RNA template; (b) utilizing thefirst DNA molecule as a template to enzymatically synthesize a secondDNA molecule thereby forming a double-stranded DNA molecule wherein thefirst DNA molecule is hybridized to the second DNA molecule; (c)utilizing the first or second DNA molecule of the double-stranded DNAmolecule as a template to enzymatically synthesize a first RNA moleculethat is complementary to either the first DNA molecule or to the secondDNA molecule; and (d) utilizing the first RNA molecule as a template toenzymatically synthesize a third DNA molecule that is complementary tothe first RNA molecule.
 51. A method for hybridizing a processed DNAsample to a population of immobilized nucleic acid molecules, the methodcomprising the step of hybridizing a processed DNA sample to apopulation of immobilized nucleic acid molecules, wherein the processedDNA sample is prepared by a method comprising the steps of: (a)utilizing an RNA template to enzymatically synthesize a first DNAmolecule that is complementary to at least 50 contiguous bases of saidRNA template; (b) utilizing the first DNA molecule as a template toenzymatically synthesize a second DNA molecule, thereby forming adouble-stranded DNA molecule wherein the first DNA molecule ishybridized to the second DNA molecule; (c) utilizing the first or secondDNA molecule of the double-stranded DNA molecule as a template toenzymatically synthesize a first RNA molecule that is complementary toeither the first DNA molecule or to the second DNA molecule; and (d)utilizing the first RNA molecule as a template to enzymaticallysynthesize a third DNA molecule that is complementary to the first RNAmolecule.